Study On Physiological,Immune Response And Ras-MAPK Signaling Pathway Of Sham-chewing In Confined Sows

In the past decades,stereotypic behavior of farm animals has been one of the most concerned animal welfare issues.In China,pregnant sows are often housed in stalls,sows are unable to express their nature and often perform stereotypic behaviors owing to the barren environment,particularly sham-chewing behavior.It is generally believed that the welfare status of sows is impaired at that time,and some scholars even speculate that sows develop negative emotions similar to depression or anxiety.However,whether the sham-chewing of sows is the external manifestation of depressionlike emotion is still lacking of verification of physiological,immune and molecular regulatory mechanisms.Therefore,in order to clarify the performance of stereotypic behaviors of pregnant sows in stalls and their relationship to serum physiological or immune levels,in addition to preliminarily explore the neural molecular regulatory mechanism of high incidence of shamchewing,a total of fifty pregnant sows(Large White × Landrace)were selected for this study,observe general behaviors(standing,sitting,lateral lying and ventral lying)and stereotypic behaviors(sham-chewing,bar-biting,trough-biting and rooting)in early,middle and late gestation(27th,62 nd and 91 st day),then measured serum physiological parameters(cortisol,Pig-MAP and CRP)and immune parameters(Ig A,Ig G,IL-6,IL-10,TNF-α and IFN-γ).Then,according to the numbers of sham-chewing observed,sows were divided into high incidence of sham-chewing(group H,n =25)and low incidence of sham-chewing(group L,n =25).After weaning,4 sows were randomly selected from group H and group L,samples of hypothalamus,hippocampus and prefrontal cortex were collected after slaughter,and hypothalamus was sequenced with transcriptome.After bioinformatics analysis,the differentially expressed genes were analyzed and enriched in GO and KEGG,then candidate genes were screened.q RT-PCR was used to verify the accuracy of the sequencing data,and Western Blot was used to detect the protein expression of key signaling pathways in the cerebral cortex-limbic system(hypothalamus,hippocampus and prefrontal cortex).Behavioral,physiological and immune results indicated that with the progress of gestation of sows,lateral lying increased significantly(p < 0.05),standing,ventral lying,sham-chewing,barbiting,trough-biting,rooting and frothy saliva score were significantly reduced(p < 0.05),sitting,standing and grooming changes were not significant(p > 0.05),and no significant changes in physiological and immune levels(p > 0.05).Sham-chewing was significantly positively correlated with serum cortisol(r = 0.314,p < 0.001),IL-6(r = 0.244,p = 0.003),IL-10(r = 0.169,p = 0.038)and negatively correlated with serum IFN-γ(r =-0.243,p = 0.003).Trough-biting was significantly positively correlated with serum cortisol(r = 0.187,p = 0.022),Ig G(r = 0.164,p = 0.045)and TNF-α(r = 0.220,p = 0.007).Rooting was significantly positively correlated with serum Pig-MAP(r =0.204,p = 0.012),Ig A(r = 0.168,p = 0.040),Ig G(r = 0.231,p = 0.004)and IL-10(r = 0.178,p =0.030).Frothy saliva score was significantly positively correlated with serum IL-10(r = 0.196,p =0.016)and negatively correlated with serum IFN-γ(r =-0.201,p = 0.014).Hypothalamic transcriptome results after grouping showed that compared with group L,1286 genes were significantly differentially expressed in group H,among which 934 genes were up-regulated and352 genes were down-regulated.The results of GO enrichment analysis showed that in biological process(BP),cell component(CC)and molecular function(MF),the most significant terms of enrichment were trans-synaptic signaling,neuron part and gated channel activity.These terms are strongly related to synaptic plasticity,synaptic transmission,neurodevelopment and neuropsychiatric diseases,FGF12,SNAP25,CAMK1 D,SYNDIG1,GABRD,RBFOX1,CRH,HCN1,HCN4,GLRA3,GRIN2 A,ANXA2,MBP and NTF4 genes with high fold change and more in-depth functional research were selected for depressive emotion-related genes.KEGG enrichment analysis results indicated that differential expressed genes were significantly enriched in 29 pathways,such as Ras and MAPK signaling pathway.The results of Western Blot revealed that,except for the relative expression of Ras protein in hypothalamus had no significant difference(p >0.05),the relative expression of Ras,p-Raf/Raf,p-MEK/MEK and p-ERK/ERK of group H were all significantly lower than those of group L(p < 0.05),suggesting that the Ras-MAPK signaling pathway in the cortex-limbic system of sows with high incidence of sham-chewing was inhibited.In conclusion,frothy saliva can be utilized as a reliable indicator for evaluating stereotypy of sows,sows with high incidence of stereotypic behaviors tried to improve stress and humoral immunity to cope with and adapt to confined environment,long-term restricted sows were in chronic stress and depression-like state.The results of hypothalamic transcriptome indicate that there is a molecular genetic basis for depression-like symptoms in sows with high incidence of sham-chewing,the 14 differential genes screened and cortex-limbic system Ras-MAPK signaling pathway might be candidate genes and key signaling pathways involved in high incidence of sham-chewing and depression-like emotion,the Ras-MAPK signaling pathway of sows with high incidence of shamchewing was inhibited.